Proteins and polypeptide hormones which bind to cell surface receptors can be internalized rapidly by receptor-mediated endocytosis. In several cases, it has been found that the receptors cluster over specialized (coated) regions of the plasma membrane which pinch off to form endocytic vesicles. This process can function both to remove occupied receptors from the cell surface and to deliver proteins to intracellular organelles. Using fluorescently labeled proteins (alpha2-macroglobulin) and hormones (insulin and epidermal growth factor) it has been possible to visualize receptor-mediated endocytosis directly. I intend to use a microscope fluorescence spectrophotometer system to obtain quantitative information about the rate and extent of receptor-mediated endocytosis. The clustering of receptor-ligand complexes over coated regions brings the ligands into close contact so that fluorescence energy transfer will occur between appropriately labeled ligands. Little energy transfer occurs outside of the clusters, so fluorescence energy transfer will be used as a monitor of the extent of the receptor clustering which precedes endocytosis. Once appropriate assay conditions have been determined, the rate and extent of receptor clustering will be determined under a variety of conditions, including growing vs. quiescent and normal vs. transformed cultured cells. The effects of various compounds which inhibit clustering will be examined to obtain information about the molecular mechanism of receptor clustering. The role of clustering in hormone action will be examined, and the possibility that there are regulatory mechanisms for controlling the rate of clustering will be investigated.